A. Field Of The Invention
This invention relates to monoclonal antibody technology. More specifically, it relates to myelomas which can be used to create human/human hybridomas.
B. Description Of The Art
The pioneering work of G. Kohler and C. Milstein, 6 Eur. J. Immunol. 511-519 (1976) (the disclosure of this article and of all other articles referred to herein are incorporated herein as if fully set forth) spawned monoclonal antibody research. The goal of this research was the development of ways to obtain virtually unlimited quantities of pure antibody with a single specificity.
Antibodies are protein molecules produced by animals to protect them against invasion by bacteria, parasites, viruses, and other foreign substances. For nearly any foreign substance ("antigen"), there are antibodies able to recognize only that chemical structure. The human body makes millions of different genetically programmed antibody molecules, each recognizing a different target or chemical structure. As normal components of our blood stream, antibodies are extremely useful to medicine, science, and industry, because of their specificity in recognizing and physically binding to substances in the human body.
Prior to the work of Kohler and Milstein, antibodies were produced in infected animals and purified from their blood. However, this method had significant limitations. The antibody quality changed with each lot. Further, even using large animals, there was a practical limitation to the quantity obtained.
More importantly, the blood of any animal contains the sought-after antibody along with millions of other antibodies, each recognizing a different structure. Thus, conventionally-produced antibodies had multiple specificities and were difficult or impossible to use for many applications.
The procedure Kohler and Milstein used for producing monoclonal antibodies began with the injection of the antigen or target chemical into a mouse. When lymphocytes began proliferating, the lymphocyte-rich spleen was taken from the mouse. Among the many lymphocytes were a few with the antibody molecules of the desired specificity. The entire population of lymphocytes obtained from the spleen was then mixed with mouse "myeloma" (bone marrow tumor) cells.
Like most cancer cells, myeloma cells can survive indefinitely when grown in cultures. Next, the myeloma cells and lymphocytes were fused, most commonly by adding polyethlyene glycol. The goal was a "hybridoma," a cell that combined the antibody producing capability of lymphocytes with the survival capability of myeloma cells. Somewhere among the hundreds of million cells treated with PEG, there were usually a few hybridomas producing the desired antibody.
These cells were normally found and separated in a three step process of selection, screening, and cloning. Selection first identifies only the properly fused hybridoma cells. This is readily achieved (because unfused lymphocytes will naturally die after a few days in culture). The remaining task in selection is to eliminate myeloma cells which have not fused with lymphocytes.
To achieve this, the original myeloma cells are usually genetically altered to lack the entire enzyme thymidine kinase (TK). When cells lacking TK are grown in a special culture fluid, HAT, they die. However, if the myeloma fuses with a normal lymphocyte, the lymphocyte provides the TK enzyme, and the hybridoma cell grows.
Once properly fused hybridomas have been selected, they are screened for the single hybridoma cell that is producing the desired antibody molecule. The hybridoma cells are grown in small colonies, each in a separate well of a culture dish. Several techniques are used to identify a hybridoma producing the right antibody. One, radioimmunoassay, is widely used. The target antigen is chemically bound to the bottoms of small wells in a plastic tray. A little culture fluid from a well containing hybridoma colonies is added to one of the antigen-containing wells. Cultures with hybridomas secreting the desired antibody can be identified because the antibody will bind to the antigen in the dish and all others will be washed away.
At this point, a radioactively labelled reagent that specifically reacts with antibody molecules (often another antibody specific for antibody proteins) is added. After an appropriate incubation, the wells are washed again. Finding the desired monoclonal colony is simply a matter of locating the radioactive wells.
The final step is to ensure that all cells in the culture really are a clonal population. Cells are usually diluted so that only one cell is deposited into a culture vessel. The single cell divides, producing a clone of identical hybridoma cells. The antibody produced by these cells is a monoclonal antibody with the desired specificity. Because the hybridoma cell line is virtually immortal, it can be used essentially forever to produce its particular antibody, in mass quantities.
Unfortunately, the vast majority of cell hybridization products now available are rodent monoclonal antibodies. These have been used to locate, quantify and purify molecules, to study molecular architectures and to diagnose and treat diseases both in vitro and in vivo. The most useful application of monoclonal antibodies promises to be the clinical treatment of humans; but here the use of rodent antibodies introduces the problem of a host immune response against the foreign protein (serum sickness). In addition, some antigens in human tissue lack immunogencity in xenogeneic systems, and many human diseases involving autoantibodies rely on the patient to make an immune response. Thus, it can be seen that a need has existed for human counterparts for rat monoclonal antibodies.
Initial attempts at generating immortal human versions involved the fusion of human lymphocytes with mouse myeloma cells. This approach was unsuccessful because the hybrids preferentially lost human chromosomes, produced mouse myeloma protein, or did not express the Ig producing genes.
Recently, this approach was improved by producing a mouse:human heteromyeloma line which is reportedly capable of forming stable "tribrid" human immune B lymphocytes. However, the problem of chromosomal exclusion with such a hybrid is still very real, and myeloma protein produced by the cell complicates purification and lowers efficiency.
A very different alternative approach, transformation of antigen-primed B lymphocytes by Epstein-Barr virus (EBV) led to some initial success, but these cultures yielded low levels of specific antibody and tended to be unstable after time in culture.
Combining approaches, several investigators fused EBV-transformed human myeloma cell lines with antigen-primed human B lymphocytes. Resulting hybrids synthesize their original myeloma protein, monoclonal antibody and permuted molecules derived from fusion partners. This is an unsatisfactory solution because of the safety problems involved in the use of Eptein-Barr virus, and the separation problems caused by the presence of the myeloma protein and the associated permuted molecules.
Simply having an ideal fusion partner is not the only obstacle to the production of human/human hybridomas. It is also important to have a source of a large number of active, immune human B lymphocytes as the gene source for the human/human hybridomas. There are two routes of immunization, namely in vivo (in the body) and in vitro (in the test tube). The former occurs naturally in pathologic conditions, during infections or by vaccinations, but normal immunizations of humans with certain interesting antigens can be ruled out due to serious ethical problems. In vitro immunization is not without its attendant problems.
In vitro immunization requires a cell source, usually a spleen, thymus, synovial fluids, gut lymphocytes, or peripheral blood lymphocytes ("PBLs"). PBLs are not very active mitotically, and only about 30% of the leukocytes from normal peripheral blood are B lymphocytes. Thus, investigators have used a mitogen such as pokeweed or lipopolysaccharide to stimulate PBLs - thereby increasing the number of functional hybrids. This resulted in some success with the IgM-producing lymphocytes, but the IgG-producing lymphocytes which are of greater interest do not respond well to this approach. Thus, the methodologies of human lymphocyte hybridization did not include a good human/human hybridoma myeloma fusion partner, or a good source of active human B cells producing IgG antibodies of any desired specificity.